Project Summary The understanding of cancer has evolved rapidly over the last decade, particularly with discoveries regarding the role of physical factors, such as extracellular matrix (ECM) stiffness and cellular forces, in carcinogenesis. This research has shown that altered ECM stiffness is not just a symptom of tumors, but is now known to trigger the actual onset of and progression of malignancy. Another key finding is that cellular traction stresses increase with increasing metastatic potential, suggesting that cell traction forces could be a biomarker for the likelihood of metastasis. Additionally, it has been found that (2D) collective behavior of cell populations can be significantly different from that of isolated cancer cells, and that cell migratory behavior in 3D matrices is significantly different migration on 2D surfaces. Although this has motivated the adoption of 3D microenvironments in cancer mechanobiology research, current imaging methods to quantify ECM mechanical properties and local cellular forces only provide 2D imaging, or when they do support 3D imaging, they do not provide long-range volumetric measurements of collective mechanical behavior with cellular resolution. The central objective of this proposal is to develop quantitative reconstruction capabilities for OCT-based techniques recently developed by the PI's group for volumetric imaging of cell traction forces and ECM mechanical properties. These new quantitative capabilities will be integrated with a fluorescence confocal microscopy module, to demonstrate a novel imaging platform with unprecedented capabilities for time-lapse imaging studies of biophysical cell-ECM interactions in 3D environments. Aim 1 will develop the capabilities for quantitative 3D reconstruction of ECM mechanical properties and validate it against rheometry and atomic force microscopy (AFM). Aim 2 will demonstrate our OCT-based imaging of 3D cell traction forces using low-density cell cultures, integrating cellular resolution imaging of ECM mechanical properties over millimeter-scale volumes. The demonstration of these novel, integrated imaging capabilities in low-density cell cultures will be followed by a demonstration in dense tumor spheroid cell cultures, where we will compare traction forces and ECM remodeling at the main spheroid boundary versus surrounding invasion strands. Aim 3 will add a confocal fluorescence imaging module to our OCT system, and we will demonstrate that this imaging platform can perform time-lapse reconstruction of 3D cell traction forces and cell-induced changes in ECM mechanical properties in a multiple-cell population migrating in 3D collagen. This will enable the first direct comparison of the time-varying traction forces of different cell types simultaneously migrating in 3D collagen. Our novel 3D imaging platform for systems mechanobiology research could lead to a deeper understanding of potential biophysical (mechanical) hallmarks of cancer, that can be used in the future to design and test new `mechano-therapies' that target/modulate the mechanical properties of the ECM.